Carbohydrates play a central role in a wide variety of normal and abnormal biological recognition processes. For example, oligosaccharides and polysaccharides, as components of glycoproteins and glycolipids, are ubiquitous on cell surfaces and function as cell-surface markers for recognition by protein receptors. Although such carbohydrate-mediated interactions are important in biological events, an understanding of the structure-activity relationships (SAR) of carbohydrates has developed very slowly because of the difficulty of synthesizing well-defined oligosaccharides and polysaccharides for study.
For example, carbohydrates on cell surfaces have been implicated in chronic inflammation, in viral and bacterial infection, and in tumorigenesis and metastasis. Inhibiting interactions between cell surface carbohydrates and their protein receptors could provide an effective means of preventing or treating various diseases. Consequently, a great potential exists for, and a great amount of research is directed to, the use of synthetic oligosaccharides and polysaccharides as therapeutic agents.
One application of synthetic oligosaccharides is inhibition of cell adhesion. Thus, there is interest in synthetic carbohydrates that interfere with selectin and/or integrin binding, and, therefore, of use in the treatment of asthma, ARDS, reperfusion injury, multiple sclerosis, and various other chronic inflammatory diseases.
Synthetic carbohydrates also can be useful to inhibit bacterial adhesion to human tissue. Certain oligosaccharides and polysaccharides also can be useful to induce immune responses, for antibody production, e.g., as vaccines, or to induce disease states in research animals.
Despite such uses of synthetic oligosaccharides and polysaccharides in research and therapeutic applications, no universally applicable method exists for the synthesis of these complex molecules presently. Enzymatic methods have been developed that are effective in the regiospecific and stereospecific formation of glycosidic linkages, but the enzymes are relatively specific for particular substrates, and are not widely applicable to the variety of oligosaccharides and polysaccharides that must be synthesized. See, O. Karthaus et al. J. Chem. Soc. Perkin Trans., 1, 1851-1857 (1994), and M. Schuster et al., J. Amer. Chem. Soc., 116, 1135 (1994).
Oligosaccharide synthesis is not limited to an enzymatic method, and, therefore, carbohydrate synthesis has become a very active field of research. Other glycosylation reactions and strategies for carbohydrate synthesis have been developed. As a result, some relatively complex oligosaccharides have been synthesized, but to date no generally applicable “universal” synthetic method that can reliably generate glycosidic linkages with control of regiochemistry and stereochemistry exists.
Persons skilled in the art are aware that subtle changes in the structures of glycosyl donors and acceptors change the regiochemical and stereochemical outcomes, and the yields, of existing glycosylation reactions. Consequently, every oligosaccharide synthesis is a unique undertaking. The state of the art, therefore, does not permit the synthesis of large libraries of oligosaccharides for screening purposes
Combinatorial synthesis has been used to identify drug leads and elucidate structure-activity relationships in specific areas of drug discovery. Combinatorial methods of synthesizing peptides and nucleic acids on solid supports to provide combinatorial libraries of peptides and nucleic acids have been available for many years. But, combinatorial methods to synthesize carbohydrates on the solid phase only now are being developed. See Liang et al., Science, 274, pp. 1520-1522 (1996). The reliable preparation of a combinatorial oligosaccharide library requires reliable and consistently If high-yielding reactions, which give well-defined products from a variety of substrates under standardized reaction conditions.
Carbohydrates are notoriously difficult to synthesize, but research groups have made advances in simplifying and accelerating the synthetic process. For example, an automated solid-phase oligosaccharide synthesizer that facilitates preparation of carbohydrates has been developed (Science, 291, 1523 (2001)). The automated oligosaccharide synthesis allows production of oligosaccharides in about 1% of the time required using previous methods.
A comprehensive carbohydrate-based combinatorial capability requires an ability to link, attach, and modify sugars. However, the execution of such a comprehensive strategy is difficult because of the complexity of carbohydrate-based systems and the synthetic difficulties related to their construction and modification.
In addition, a simple monosaccharide contains multiple reactive sites and an anomeric center with difficult-to-control stereochemistry. Traditionally, a site selective reaction is controlled by complicated protecting group schemes. Further, the construction of multiple consecutive glycosidic linkages or glycosidic linkages to differing substrates typically relied upon different glycosylating reagents because of the lack of a single general glycosidic bond-forming reaction reliably applicable to a wide variety of substrates. Therefore, the success of a carbohydrate-based combinatorial program depends on effectively solving the unique chemical problems posed by carbohydrates.
The linking of monosaccharide units through a glycosidic bond is fundamental to the synthesis of oligosaccharides and various glycoconjugates. Therefore, the development of carbohydrate-based technology requires the construction of glycosidic bonds in the solid or liquid phase. The formation of glycosidic bonds is central to the field of glycobiology and to the preparation of a carbohydrate-based therapeutic or diagnostic agent.
Thioglycosides are among the most widely used glycosyl donors because of their ease of preparation and shelf stability. Thioglycosides also are compatible with numerous protection and deprotection synthetic steps, as well as being orthogonal to several other glycosidic bond-forming reactions, such as the trichloroacetimidate and glycosyl fluoride methods. See, P. J. Garegg, Carbohydr. Chem. Biochem., 52, pp. 179-266 (1997).
A thioglycoside requires activation to form a glycosidic bond. However, the stability that renders thioglycosides attractive as a donor, sufficiently reduces their reactivity in coupling reactions such that reaction times often are very long (e.g., up to ten days). Current methods of activating thioglycosides also suffer from one or more other disadvantages, such as unstable or unavailable activating agents, the need to oxidize the thioglycoside to the sulfoxide, poor stereoselectivity, and lack of generality. In addition, expensive reagents, which typically suffer from stability problems, are required for activation. Frequently, reagents used to activate thioglycosides are based on heavy (e.g., mercury) or precious (e.g., silver) metals, and pose problems of expense, environmental and toxicological concerns, and/or disposal.
Nonmetallic thiophiles of varying stability and activity currently in common use include dimethyl(methylthio)sulfonium triflate (DMTST), methylsulfenyl triflate (MeSOTf), benzeneselenyl triflate (PhSeOTf), iodonium dicollidine perchlorate (IDCP), and N-iodosuccinimide-trifluoromethane sulfonic acid (NIS/TfOH). Kahne's sulfoxide glycosylation method (D. Kahne et al., J. Am. Chem. Soc., 111, 6881-6882 (1989)) and related methods using glycosyl sulfimides (S. Cassel et al., Tetrahedron Letters, 39, 5175-5178 (1998)) are distinct from the mainstream methods because these methods require prior oxidation of the thioglycoside but, in doing so, permit the formation of highly reactive glycosylating species at low temperature.
Investigators, therefore, are searching for improved methods of activating thioglycosides, and forming of glycosidic bonds in general. One research group has developed a polymer-supported synthesis of oligosaccharides (O. J. Plante et al., Science, 291. pp. 1523-1527 (2001)). However, this technology does not permit the preparation of β-mannosides, and also utilizes a very unstable glycosyl phosphate and/or trichloroacetimidate.
The present invention provides a method of forming glycosidic bonds utilizing thioglycosides and an activating system that overcomes problems associated with prior methods of forming glycosidic bonds, and permits the automated synthesis of oligosaccharides and polysaccharides.